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| Status |
Public on Mar 24, 2011 |
| Title |
K562 RiboMinus Random Rep1 P5 8-21-2009 fc1.ch14 Reads5_8_21fc1ch14 |
| Sample type |
SRA |
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| Source name |
K562
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| Organism |
Homo sapiens |
| Characteristics |
rna type: RiboMinus
cdna primer: Random
comment: Rep1
cell line: K562
replicate: Rep1
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| Growth protocol |
Liver (Clontech), brain (Clontech), and K562 (Ambion) RNAs were purchased from commercial sources. HL60 RNA was prepared by MIR Preclinical Services from cells grown under standard conditions or treated with 0.1% DMSO and 0.1uM t-retenoic acid for 5 days.
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| Extracted molecule |
total RNA |
| Extraction protocol |
Before further fractionation, total RNA was treated with DNase I as follows: 50μg of total RNA was mixed with 10μl of 10X DNase I buffer (Roche), 2μl of RNaseOut (Invitrogen) and 8μl of recombinant DNase I (10U/μl, Roche) and incubated for 45 minutes at 37°C. The RNA was then purified using the RNeasy MinElute kit (Invitrogen). The DNase I-treated total RNA was either unfractionated (total RNA) or fractionated using one of the following methods: 1. Depleted of ribosomal RNA (rRNA) using the RiboMinus kit (Invitrogen) 2. polyA fraction was selected using a magnetic bead-based purification kit (Dynabeads mRNA purification kit, Invitrogen) or, 3. polyA fraction was selected using the oligo-dT cellulose method (Micro Poly(A)Purist Kit, Ambion). Some RNA samples were fragmented by heating at 95C for 10 min prior to cDNA synthesis. 100-400 ng of DNase I -treated RNA, except where noted, was mixed with the following reagents from the SuperScript III kit (Invitrogen). First 10μl of 50ng/μl Random Hexamers and 2μl of 10mM dNTPs were added in the total volume of 25μl. When employing selected primers, the same conditions were used. The mixture was then placed in a thermocycler and heat denatured at 65°C for 5 min followed by rapid cooling on ice. Next, 5μl of 10X cDNA synthesis buffer, 5μl of 0.1M DTT and 10μl of 25mM MgCl2 were added. The samples were returned to the thermocycler and allowed to incubate at 15°C for 20 min. Then, 2.5μl of RNaseOut and 2.5μl of SuperScript III reverse transcriptase were added and the samples were incubated at 25°C for 10 min, 42°C for 40 min, 55°C for 50 min and 70°C for 10 min. After reverse transcription, RNA was removed by adding 1μl of RNaseH (Invitrogen) and 1μl of RNase If (New England BioLabs) to each sample and incubating at 37°C for 30 min. The cDNA was then purified by two rounds of purification over Performa columns (EdgeBio) and quantified using a NanoDrop spectrophotometer. Next, a 3â poly-A tail was added to the cDNA samples. cDNA (100ng) was mixed with a control oligo to monitor tail length and water in a total volume of 33.5μl. The mixture was denatured at 95°C for 5 min followed by rapid cooling on ice. 5μl of 2.5mM CoCl2, 5μl of 10X terminal transferase (TdT) buffer (New England BioLabs), 5 µl of 50µM dATP and 1.5μl of TdT (20U/μl, New England BioLabs) was then added and the samples were incubated at 42°C for 1 hr, and at 70°C for 10 min. The 3â ends of the polyA-tailed cDNA were then blocked with biotin-ddATP. The sample was denatured at 95°C for 5 min followed by rapid cooling on ice. 0.3µl of 1mM biotin-ddATP (Perkin Elmer) and 1.5µl of TdT were added followed by incubation at 37°C for 45 min and 70°C for 10 min.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
other |
| Instrument model |
Helicos HeliScope |
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| Description |
Reads counts with no normalization
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| Data processing |
Reads were aligned to UCSC gene tracks using IndexDPgenomic (hg18) with a cutoff score of 4.3. Reads with multiple equivalent alignments were fractionally distributed among the various genes. Reads aligning to ribosomal and mitochondrial transcripts were discarded. When channels were added together, it was done prior to any normalization. Aligned reads were normalized to reads per million for comparison. No length correction was performed.
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| Submission date |
Mar 23, 2011 |
| Last update date |
Jun 11, 2013 |
| Contact name |
John F Thompson |
| Organization name |
Helicos BioSciences
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| Department |
Genomic Sciences
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| Lab |
Thompson
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| Street address |
One Kendall Square
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| City |
Cambridge |
| State/province |
MA |
| ZIP/Postal code |
02139 |
| Country |
USA |
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| Platform ID |
GPL14761 |
| Series (1) |
| GSE28123 |
Protocol Dependence of Sequencing-based Gene Expression Measurements |
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| Relations |
| BioSample |
SAMN02196819 |
| Supplementary data files not provided |
| Processed data are available on Series record |
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